In his classic work The Specificity of Serological Reactions published in 1936, Landsteiner showed that antibody formation against a simple azo compound could be induced by coupling the compound to an immunogenic molecule such as serum protein. Although the simple azo molecule would not elicit antibody formation when injected alone, the azo-protein conjugate elicited the production of anti-azo antibodies as well as anti-protein antibodies when injected into animals. The notion that the small nonimmunogenic molecule was using the large immunogenic protein molecule as a carrier was thus developed. The molecule which derived immunogenicity by being conjugated to the carrier was termed the hapten.
The phenomenon of a relatively large molecule potentiating the immunogenicity of a small molecular entity to which it is attached is known in the art as the "carrier effect".
Carrier effects can be defined as immunity to one determinant (the `helper` or T cell determinant) of a multideterminant immunogen enhancing the response to another determinant (the B cell determinant). Thus, T cells, by recognizing helper determinants on the antigen somehow help B cells to make antibody against the B cell specific antigen. Furthermore, it is now known that carrier effects are not confined to hapten-protein conjugates and can be demonstrated, for example, with subunits on protein molecules. Rajewsky et al., J. Exp. Med. 126:581 (1967).
It is now well established that most antigens require T-cell help to induce B cells to produce antibodies. Classic cognate T cell help requires direct T cell-B cell interaction mediated by an antigen bridge. According to Rajewsky et al., J. Exp. Med., 129:1131 (1969), the antigenic determinants recognized by T helper cell (Th) and B cells must be covalently linked to form a single molecular entity. More recently, several groups have suggested that covalent linkage may not be necessary when the T cell determinant and B cell determinant are presented in the context of a large particle, such as a virus. This mechanism of T cell help has been termed intermolecular/intrastructural because the T and B cell determinants are present on molecular entities that are not covalently linked but are operatively linked in terms of T and B cell interaction because they are within a structure the immune system sees as being unitary. See, Russel et al., Nature, 280:147 (1979), Lamb et al., J. Immunol., 129:1465 (1982), Scherle et al., J. Exp. Med., 164:1114 (1986), and Lake et al., Cold Spring Harbor Symp. Quant. Biol., 41:589 (1976). Some, however, can induce antibody formation in the absence of T-cells. These are called thymus-independent (T-independent) antigens. Most, if not all, antigens which act in the absence of helper T-cells are composed of repeating subunits of the same antigenic determinant. The most commonly used T-independent antigens are polymerized flagellin (a protein of repeating subunits) or various polysaccharides (which are repeating units of sugars).
Hapten-carrier conjugates are widely used today in determining the function of the various cells in the immune response. However, while hapten-carrier conjugates have served the research community well in its investigations of the nature of the immune response, they have not yet been of significant use in the production of immunogens that play a role in diagnostic or therapeutic modalities.
Recently, it has been determined that a pathogen related protein can be immunologically mimicked by the production of a synthetic polypeptide whose sequence corresponds to that of a determinant domain of the pathogen related protein. Such findings are reported by Sutcliffe et al., Nature, 287:801 (1980), and Lerner et al., Proc. Natl. Acad. Sci. USA, 78:3403 (1981).
Moreover, Gerin et al., (1983) Proc. Natl. Acad. Sci. USA, 80:2365 have recently shown limited protection of chimpanzees from hepatitis B virus upon immunization with carrier-bound synthetic polypeptides having amino acid residue sequences that correspond to the sequence of a determinant portion of HBsAg; in particular, residues 110-137 of the "S" (surface) region. However, the carrier protein used in these studies was keyhole limpet hemocyanon (KLH), a T cell dependent carrier that is not fit for use in humans because it is a source of irritation that leads to severe inflammation.
The art has long sought a T cell stimulating carrier protein capable of enhancing the immunogenicity of polypeptide immunogens that does not produce unacceptable side effects in human subjects. Immunogenic natural proteins, particularly tetanus toxoid, have been used most frequently when a carrier suitable for human administration was needed. However, even the use of tetanus toxoid as a carrier has been restricted due to problems with dosage limitations and risk of sensitization to the toxoid itself. In addition, an epitopic specific suppression can occur even in individuals already immunized against tetanus.
Vigorous efforts in pursuit of identifying highly immunogenic carrier proteins have resulted in Delpeyroux et al., Science, 233:472-475 (1986) reporting the use of the HBV surface protein (S protein) as a carrier for a poliovirus polypeptide immunogen. Those investigators constructed a recombinant deoxyribonucleic acid (DNA) protein expression vehicle that produces a fusion protein, designated HBsPolioAg, capable of forming particles closely resembling authentic 22-nanometer HBsAg particles. HBsPolioAg consists of HBV S protein having an 11 amino acid residue sequence insert corresponding to amino acids 93-103 of capsid protein VPI of poliovirus type 1 (Mahaney strain).
Delpeyroux et al. reported that antisera from mice immunized with HBsPolioAg had a significant titer of poliovirus neutralizing antibodies. However, they also reported that the "best titer obtained was low by poliovirus standards and further work to improve the titer is needed".